Neosynthesis and Activation of Rho by Escherichia coli Cytotoxic Necrotizing Factor (CNF1) Reverse Cytopathic Effects of ADP-ribosylated Rho

Abstract

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln 63 , caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30 -60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.The low molecular mass GTPases of the Rho family (Rho, Rac, and Cdc42) play key roles in the control of the actin cytoskeleton (1). In several cell lines, activation of RhoA induces formation of stress fibers and focal adhesions (2), whereas Rac and Cdc42 cause the formation of lamellipodia and filopodia, respectively (2-4). Moreover, Rho GTPases act as molecular switches in various signaling processes (5) including secretion (6), phagocytosis (7), endocytosis (8, 9), cell cycle progression (10), transcriptional activation (11) (22). C3 exoenzyme in particular is widely used as a cell biological tool to study the functions of Rho (23).Inasmuch as C3 exoenzyme appears to enter cells via nonspecific pinocytosis, its cell accessibility is poor (24). Recently, we constructed a C3 fusion toxin, which enters cells by using the binary C. botulinum C2 toxin as a carrier (25). The actin-ADP-ribosylating C2 toxin consists of the ϳ80-kDa binding component (C2II) and the 49-kDa enzymatic component (C2I) (26 -28). Both C2 components are separate proteins which assemble at the cell surface after C2II has bound to an unknown receptor. The C3 fusion toxin (C2IN-C3) contains the N-terminal domain (amino acid residues 1-225) of C2I (C2IN), which has no enzyme activity but binds to the C2II component and is sufficient for translocation of C2I into the cytosol. In contrast to C3 exoenzyme, the C2IN-C3 fusion toxin enters cells readily and causes the depolymerization of stress fibers at lo...

Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 Exoenzyme in the Rho-Guanine Nucleotide Dissociation Inhibitor-1 Complex

“…through prevention of its membrane binding. In a recombinant system, ADP-ribosylation reduced Lbc-catalyzed GTP loading of Rho (22). Both effects together may prevent GTP loading of ADP-ribosylated Rho.…”

“…However, direct inhibition of Rho-effector coupling is obviously not the mode of action for how Rho is inactivated by ADP-ribosylation in intact cells. The notion that ADP-ribosylated Rho is biologically active is further supported by the observation that sequential treatment of cells with C3 followed by CNF1 results in typical features of the CNF1 morphology (22,24). ADP-ribosylated RhoA Q63E , likely loaded with cellular GTP, is obviously active, i.e.…”

“…The disadvantage of poor cell accessibility because of failure of receptor binding and translocation domain has been overcome by different approaches: 1) microinjection of C3 (14); 2) electroporation of cells in the presence of C3 (15); 3) permeabilization by digitonin (16) or streptolysin O (17); 4) intracellular expression of C3 (18 -20); 5) using chimeric toxins that recruit the cell entry machinery of other toxins, C3 is fused to enzyme-deficient diphtheria toxin or C. botulinum C2 toxin (21)(22)(23). The latter possibility, a chimera with the binary C. botulinum C2 toxin, was applied in this study.…”

“…It has been reported that the typical feature of the disrupted actin filament system caused by C3-catalyzed ADP-ribosylation is reversed by the cytotoxic necrotizing factor (CNF) (22,24) that is known to transform Rho constitutively active by deamidation of glutamine 63 (25,26). Because deamidation merely blocks GTP hydrolysis, ADP-ribosylated Rho is able to interact functionally with its effectors.…”

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…However, inactivation of Rho by ADPribosylation can be overcome by treatment with the cytotoxic necrotizing factor CNF1. CNF1 deamidates RhoA at RhoA Q63 , which is located in the switch II region, and thereby constitutively activates RhoA (Barth et al 1999). In contrast, no evidence was found for the hypothesis that ADP-ribosylation prevents binding and activation of downstream effectors (Sehr et al 1998;Genth et al 2003b) or influences the GAP-mediated Rho inactivation (Sehr et al 1998).…”